Detection of NO by using guanylyl cyclase and cGMP production as a readout system

ABSTRACT

The present invention relates to a method for determining NO enzymatically and its use for the identification of substances which can modulate a nitric oxide synthase activity.

RELATED APPLICATIONS

Benefit of U.S. Provisional Application Ser. No. 60/529,203, filed onDec. 12, 2003 and of European Patent Application 03 027 159, filed onNov. 26, 2003 are hereby claimed, and which applications areincorporated herein in their entireties.

INTRODUCTION

The L-arginine-nitric oxide pathway is involved in a large variety ofphysiological processes (Mayer; B. et al., 1997; Vallance, P. et al.,2002; Wendy, K. et al., 2001), like for example the regulation of bloodpressure, platelet aggregation or host defence against pathogens. Nitricoxide (NO) can be for example produced by nitric oxide synthase (NOS)representing a group of enzymes existing as three different isoforms ofNOS: eNOS, nNOS and iNOS (Janssens, S. P. et al., 1992; Marsden, P. A.et al., 1992; Nakane, M. et al., 1993; Geller, D. A. et al., 1993;Sherman, P. A. et al. 1993; Charles, I. G. et al., 1993; Maier, R. etal., 1994; Chartrain, N. A. et al., 1994).

To influence diseases which are associated with NOS activity (e.g.chronic obstructive pulmonary disease (COPD), arteriosclerosis,wound-heeling) modulation of NOS activity is wanted. Therefore, thereexists a need for substances which can modulate NOS activity.Subsequently, there is a need for e.g. an ultra high throughputcompatible assay which is sensitive enough to determine whether asubstance can modulated NOS activity.

In the following the known reaction of NOS with Arginine in presence ofNADPH, FAD, FMN and biopterin resulting in Citrulline and No and NADP⁺is given (Griffith, O. W. et al, 1995; Mayer, B. et al.; 1995; Stuehr,D. J. et al., 1991; Klatt, P. et al., 1993; Feldmann, P. L. et al, 1993;Marletta, M. A., et al., 1994; Pufahl, R. A. et al., 1993; Clement, B.et al., 1993):

To verify whether NOS activity indeed has been altered it is essentialto measure NOS activity. To measure the activity of a NOS enzyme it isnecessary to detect arginine consumption, citrulline production or NOgeneration.

Currently, only assay systems which are not compatible to miniaturizedhigh through put screening systems are known. Only radioactive systemsfor measuring arginine or citrulline are available. To make mattersworse, said systems are only meaningful at high doses of arginine orcitrulline and therewith respective miniaturising is not successfullypossible.

Other systems are not usable for a screening campaign since a substanceto be tested interacts unspecifically with substances of the system(Powell D. and Williams D., 2002).

Since NO is only stable for a few seconds it is not regarded as asuitable read-out. Commercially available NO detection systems have beenfailed as a suitable readout technique in high throughput screening(Misko T. P. et al. 1993).

For a high throughput compatible NOS activity assay for a drug screeningcampaign it is necessary to have a sensitive and miniaturizable assayformat.

DESCRIPTION OF THE INVENTION

Until now nitric oxide (NO) was regarded as a not suitable readout sinceit is only stable for a few seconds. The present invention provides fora method for detecting NO which is surprisingly accessible to a newdetection method of the present invention.

The guanylyl cyclase is a physiological target for NO. Guanylyl cyclasebecomes activated by NO several hundred fold. In the presence of GTP asaid activated guanylyl cyclase catalyses the generation of cGMP.Therewith, NO causes the generation of cGMP. As disclosed in the presentinvention said signal transduction can be surprisingly used in aninverse direction to detect NO quantitatively.

We have established a high throughput compatible assay format for thedetection of Nitric oxide synthase (NOS) activity employing guanylylcyclase and cGMP as an amplification and readout system for thedetection of NO. Our assay setup is as follows: recombinantly expressedNOS enzyme, purified NOS enzyme from a natural source or working with acell based assay system containing NOS is used to generate NO. Addingsoluble guanylyl cyclase to the system leads to an increased activationof the guanylyl cyclase enzyme. This results in the generation of cyclicGMP which is then detected using a cGMP detection system. As a cGMPdetection system e.g. commercially available detection systems can beused.

This detection system for NO is quite sensitive since the activation ofthe guanylyl cyclase results in an cGMP increase. One activated guanylylcyclase can generate several cGMP molecules therefore we have anamplification effect and can overcome the sensitivity issue.

The present invention provides therewith a technical basis for a longfelt need: An assay for determining whether a substance can modulate NOSactivity. Therewith the invention also provides for a high throughputscreening (HTS) compatible NOS activity assay for a drug screeningcampaign.

Nitric oxide synthase (NOS) according to the present invention is anyenzyme which is selected from a group consisting of: i-NOS (inducible NOsynthase, II) e-NOS (endothelial NO synthase, III), and n-NOS (neuronalNO synthase, I).

Nitric oxide synthase (NOS) activity according to the present inventionis an activity which catalyzes the formation of NO by convertingarginine to citrulline with the concomitant release of NO. NOS activityaccording to the present invention includes any further conversion ofany other substrate resulting in the generation of NO.

A substrate according to the invention is every substance which can betransformed by NOS in any other substance as long as NO is produced. Apreferred substance of the invention is arginine.

A substance to be tested to be a modulator of NOS according to theinvention is any small chemical molecule, peptide, or antibody.

A modulator according to the invention is either an inhibitor of theinvention or an activator of the invention.

An inhibitor of the nitric oxide synthase activity according to thepresent invention is identifiable by an reduced level of cGMP in amethod according to the invention which can be measured in the solutionto which the substance to be tested (in step (c)) was added.

An activator of the nitric oxide synthase activity according to thepresent invention is identifiable by an enhanced level of cGMP in amethod according to the invention which can be measured in the solutionto which the substance to be tested (in step (c)) was added.

A method according to the invention is, wherein the method comprises:

-   -   providing a solution comprising NO;    -   adding guanylyl cyclase and guanosin triphosphate (GTP) under        conditions which allow synthesis of cyclic guanosin        monophosphate (cGMP);    -   measuring cGMP. Measuring according to the invention is any        suitable detecting method which is useful of determining cGMP        quantitatively. A preferred method for measuring cGMP according        to the invention uses an antibody which is capable of binding to        cGMP.

Said method can be used to detect NO in solution.

Said method can additionally be used in a high throughput screening.Therefore, the present invention provides for a method for detection ofnitric oxide (NO)) in an high throughput (HTS) format wherein the abovementioned method is performed. Said method can be performed by using aplate having at least 96 wells, more preferred is the use of a 384 wellplate or a 1536 well plate. Also preferred is the use of a chip asreaction and/or readout platform.

In one embodiment the present invention provides for a method formeasuring nitric oxide synthase activity, wherein the method comprises:

-   -   (a) providing a solution comprising a nitric oxide synthase and        a suitable substrate under conditions which allow synthesis of        nitric oxide;    -   (b) adding guanylyl cyclase and GTP under conditions which allow        synthesis of cGMP;    -   (c) measuring cGMP.

Measuring cGMP according to the invention is any suitable detectingmethod which is useful of determining cGMP quantitatively. A preferredmethod for measuring cGMP according to the invention uses an antibodywhich is capable of binding to cGMP. Said method can be used in a highthroughput screening. Therefore, the present invention provides for amethod for measuring nitric oxide synthase (NOS) activity in an highthroughput (HTS) format wherein the above mentioned method is performed.Said method can be performed by using a plate having at least 96 wells,more preferred is the use of a 384 well plate or a 1536 well plate. Alsopreferred is the use of a chip as reaction and/or readout platform.

In a further embodiment the present invention provides for a method fordetermining whether a substance is modulator of nitric oxide synthaseactivity according to the invention, wherein the method comprises:

-   -   (a) providing a first solution comprising a nitric oxide        synthase and a suitable substrate under conditions which allow        synthesis of nitric oxide;    -   (b) providing a second solution comprising a nitric oxide        synthase and a suitable substrate under conditions which allow        synthesis of nitric oxide;    -   (c) adding a substance to be tested to the first or the second        solution;    -   (d) adding guanylyl cyclase and GTP under conditions which allow        synthesis of cGMP to the first and the second solution;    -   (e) measuring cGMP in the first and the second solution;    -   (f) comparing cGMP levels of the first and the second solution,        wherein a modulator of a nitric oxide synthase activity is        identified by different levels of cGMP.

Measuring cGMP according to the invention is any suitable detectingmethod which is useful of determining cGMP quantitatively. A preferredmethod for measuring cGMP according to the invention uses an antibodywhich is capable of binding to cGMP. Said method can be used in a highthroughput screening. Therefore the present invention provides for amethod for determining whether a substance is modulator of nitric oxidesynthase (NOS) activity in an high throughput (HTS) format wherein theabove mentioned method is performed. Said method can be performed byusing a plate having at least 96 wells, more preferred is the use of a384 well plate or a 1536 well plate. Also preferred is the use of a chipas reaction and/or readout platform.

In a further embodiment all above mentioned methods of to the inventionadditionally comprise superoxid dismutase (SOD) which can be addedtogether with guanylyl cyclase and GTP.

All methods of the present invention can be used in a high throughputscreening assays.

The present invention also provides for a kit for determining whether asubstance is a modulator—i.e. an activator or an inhibitor—of NOSactivity comprising: guanylyl cyclase, GTP and a system for detection ofcGMP. Said system is any suitable detecting method which is useful ofdetermining cGMP quantitatively. A preferred method for measuring cGMPaccording to the invention uses an antibody which is capable of bindingto cGMP.

In a more preferred embodiment said kit of the invention furthercomprises Superoxide dismutase (SOD).

In a most preferred embodiment said kit of the invention furthercomprises nitric oxid synthase (NOS) which is selected from a groupconsisting of: i-NOS, e-NOS, and n-NOS. This kit can additionallycomprise Superoxide dismutase (SOD).

In a further embodiment the present invention also provides for a kitfor measuring nitric oxide synthase (NOS) activity comprising: guanylylcyclase, GTP and a system for detection of cGMP. Said system is anysuitable detecting method which is useful of determining cGMPquantitatively. A preferred method for measuring cGMP according to theinvention uses an antibody which is capable of binding to cGMP.

In a more preferred embodiment said kit of the invention furthercomprises Superoxide dismutase (SOD).

In a most preferred embodiment said kit of the invention furthercomprises nitric oxid synthase (NOS) which is selected from a groupconsisting of: i-NOS, e-NOS, and n-NOS. This kit can additionallycomprise Superoxide dismutase (SOD).

The present invention additionally provides for a further kit fordetection of nitric oxid (NO) comprising: guanylyl cyclase, GTP and asystem for detection of cGMP. Said system is any suitable detectingmethod which is useful of determining cGMP quantitatively. A preferredmethod for measuring cGMP according to the invention uses an antibodywhich is capable of binding to cGMP.

In a more preferred embodiment said kit of the invention furthercomprises Superoxide dismutase (SOD).

The following Examples are meant to illustrate the present invention,however, shall not be construed as limitation. However, the Examplesdescribe most preferred embodiments of the invention.

EXAMPLES Example 1 Assay Description

Detection of NOS activity by using the activation of soluble guanylylcyclase, an enzyme which is well known for years (Gerzer R., et al.1981; Zabel U., et al., 1998; Hoenicka, M. et al., 1999; Lee, Y. C. etal., 2000; Kosarikov, D. N. et al.; 2001). NO produced by the differentNOS enzymes can activate soluble guanylyl cyclase and also its membranestanding counterpart. In said type of assays recombinantly expressedNOS, a cell-lysate containing NOS as well as NOS purified from a naturalsource can be used (Janssens, S. P. et al., 1992; Marsden, P. A. et al.,1992; Nakane, M. et al., 1993; Geller, D. A. et al., 1993; Sherman, P.A. et al. 1993; Charles, I. G. et al., 1993; Maier, R. et al., 1994;Chartrain, N. A. et al., 1994).

Here we describe a non radioactive HTS compatible assay format which canbe used to detect the NO produced by the NOS activity via activation ofguanylyl cyclase and subsequent detection of the produced cGMP using theAlphaScreen technology in a mix and measure mode.

Arginine as a substrate is converted by each of the three different NOSenzymes respectively, into citrulline. During this process NO is alsoproduced. Therefore NO detection can be used to monitor directly theactivity NOS enzymes. It is known that NO can directly stimulate thesoluble guanylyl cyclase. This activation leads to an increase in cGMPproduction by the activated guanylyl cyclase. The produced cGMP can bedetected and quantified. A variety of different technologies can beemployed for the detection and quantification of the newly producedcGMP. This technologies can be e.g. based on HTRF (Claret E. J., et al.,2004) or radioactivity (Perkin Elmer: Flashplate cyclic cGMP[¹²⁵I]-assay product no: SMP002J001PK) or like it is shown here based onthe AlphaScreen technology. Using the Alphascreen cGMP competition assaya biotinylated cGMP is used to generate a fluorescence signal. Thebiotinylated cGMP is binding to the AlphaScreen donor bead viastreptavidin and is binding to the AlphaScreen acceptor bead via ananti-cGMP specific antibody bound to the bead surface. If cGMP isfreshly produced by the stimulated guanylyl cyclase it is competing withthe binding to the anti-cGMP antibody. Therefore, the standardfluorescence signal is getting reduced by freshly produced cGMP, whichis not biotinylated.

Example 2 Standard Operating Procedure for Performing a Mix and MeasureHTS Compatible NOS Activity Assay

Method

In 384-well plates, 3 μl of a test compound diluted in distilled water(final concentration of compounds 5 μg/ml; DMSO 1%) is mixed with 3 μlenzyme mix dissolved in assay buffer (i-NOS final 2-10 μg/ml or e-NOSfinal 2-10 μg/ml or n-NOS final 2-10 μg/ml, sGC final 1:800 000—givenamounts of respective NOS enzymes are based on total concentration ofcell culture supernatant employed (insect cell recombinant expressionsystem)). After an incubation time of 30 minutes 3 μl substrate mixdissolved in assay buffer (GTP final 100 μM, Arginine final 3 μM, NADPHfinal 500 μM, for n-NOS 1 μM Ca²⁺ and 1 μM Calmodulin is necessary) isadded. After another incubation time of 90 minutes the reaction isstopped with 10 μl acceptor and donor bead-antibody mix dissolved instop/detection buffer (final concentration of each bead-type 15 μg/ml;antibody final 1:15000).

After an overnight incubation, the assay is measured using e.g. anAlphaquest reader (Perkin Elmer), which is measuring fluorescence at520-620 nm.

-   Assay buffer: 25 mM Tris pH 7.4, 3 mM MgCl₂, 3 mM DTT, 0.05% BSA, 3    μM BH₄, 1M FAD, 1M FMN, 10 μM    4-{[3′,4′-(Methylenedioxy)benzyl]amino}-6-methoxyquinazoline-   Stop/detection buffer: 50 mM Tris, 50 mM EDTA pH 7.4, 0.10% BSA,    0.10% Tween-20

Each assay microtiter plate contains wells with vehicle control insteadof compound (1% DMSO in water) as reference for non-inhibited enzymeactivity (100% CTL; high values) and wells with 100 μM AMT as referencefor inhibited enzyme activity (0% CTL; low values).

Each assay micro-titer plate contains a cGMP standard curve (1, 10, 100,1000, 3000, 10000, 100000, 1000000 pM/I) in duplicate used as referenceto calculate the amount of cGMP produced in every well.

The analysis of the data is performed by the calculation of thepercentage of cGMP in the presence of the test compound compared to cGMPgenerated from the positive control:

(cGMP(sample)−cGMP (low value))×100/(cGMP (high value)−cGMP (low value))

An inhibitor of the enzyme activity will give values between 100% Ctl(no inhibition) and 0% Ctl (complete inhibition).

Materials used are well known in the art, however, in the followingrespective suppliers are given: The 384 low volume plates were purchasedfrom Greiner (white; cat.-no. 784075). The AlphaScreen IgG Detection Kit(cat.-no. 6760617) and the biotinylated cGMP were bought from PerkinElmer. The anti-cGMP antibody (cat.-no. 970161) is from MerckBiosciences. The different NOS enzymes i-NOS (cat.-no. 201-069), e-NOS(cat.-no. 201-070) and n-NOS (cat.-no. 201-068), sGC (cat.-no. 201-177)and the L-Arginine (cat.-no. 101-004) were from Alexis. The NADPH(cat.-no. N-6005) and the GTP (cat.-no. G-5884) were purchased fromSigma. All other materials were of highest grade commercially available.

Example 3 Standard Operating Procedure for Performing a Mix and MeasureHTS Compatible NOS Activity Assay Optimized by Adding a Further Enzymeto the Reaction Mixture the Superoxide Dismutase (SOD)

In the following a further variant of the assay of the invention isdisclosed in which an enzyme has been added to the reaction mixture. TheSuperoxide dismutase (SOD) (Keele Jr. B. B. et al., 1970; Beckman J. S.et al., 1990) increases the sensitivity of the assay system andtherefore reduces the amount of NOS enzyme needed for performing theassay.

Method

In the 384-well plates, 3 μl of the test compounds diluted in distilledwater (final concentration of compounds 5 μg/ml; DMSO 1%) is mixed with3 μl enzyme mix dissolved in assay buffer (i-NOS final 2 μg/ml, e-NOS 5μg/ml, n-NOs 0.4 μg/ml, SOD final 300 nM, sGC final 1:800 000—givenamounts of respective NOS enzymes are based on total concentration ofcell culture supernatant employed (insect cell recombinant expressionsystem)). After an incubation time of 30 minutes 31 μl substrate mixdissolved in assay buffer (GTP final 100 μM, Arginine final 3 μM, NADPHfinal 500 μM, for nNOS 1 μM Ca²⁺ and 1 μM Calmodulin is necessary) isadded. After another incubation time of 90 minutes the reaction isstopped with 10 μl acceptor and donor bead-antibody mix dissolved instop/detection buffer (final concentration of each bead-type 15 μg/ml;antibody final 1:15000).

After an overnight incubation, the assay is measured using e.g. anAlphaquest (as outlined in Example 2).

-   Assay buffer: 25 mM Tris pH 7.4, 3 mM MgCl₂, 3 mM DTT, 0.05% BSA, 3    μM BH₄, 1 μM FAD, 1 μM FMN, 10 μM    4-{[3′,4′-(Methylenedioxy)benzyl]amino}-6-methoxyquinazoline-   Stop/detection buffer: 50 mM Tris, 50 mM EDTA pH 7.4, 0.10% BSA,    0.10% Tween-20

Each assay microtiter plate contains wells with vehicle control insteadof compound (1% DMSO in water) as reference for non-inhibited enzymeactivity (100% CTL; high values) and wells with 100 μM AMT as referencefor inhibited enzyme activity (0% CTL; low values).

Each assay microtiter plate contains a cGMP standard curve (1, 10, 100,1000, 3000, 10000, 100000, 1000000 pM/l) in duplicate used as referenceto calculate the amount of cGMP produced in every well.

The analysis of the data is performed by the calculation of thepercentage of cGMP in the presence of the test compound compared to cGMPgenerated from the positive control.

(cGMP(sample)−cGMP (low value))×100/(cGMP (high value)−cGMP (low value))

An inhibitor of the enzyme activity will give values between 100% Ctl(no inhibition) and 0% Ctl (complete inhibition).

Example 4 Stimulation of Soluble Guanylyl Cyclase and Subsequent cGMPDetection

The principle mechanism of the stimulation of the soluble guanylylcyclase can be shown by using a NO donor compound like for example DEANONOate or others like DETA NONOate, DPTA NONOate, GEA 5024 or GEA 3162(Drago R. S. & Paulik F. E., 1960; Drago R. S. & B. R. Karstetter B. R.1961; Maragos, C. M. et al, 1991; Wink D. A., et al. 1991; Hrabie, etal. 1993; Robak, et al., 1992; Moilanen, E. et al. 1993; Corell, T. etal. 1994; Karup, G. et al. 1994; Malo-Ranta, U. et al. 1994; Ma H. T. etal. 1999). These type of compounds produce NO which can stimulate thesoluble guanylyl cyclase which leads to a cGMP production. FIG. 1 showsthe cGMP production after stimulating the soluble guanylyl cyclase withDEA NONOate in a dose dependent manner. For detection of cGMP anAlphaScreen competition assay was used.

Example 5 Inhibition of Different NOS Enzymes Using Standard Inhibitors

AMT ODQ L-NMMA 1400W L-NIL enzyme [μM] [μM] [μM] [μM] [μM] iNOS 1.3 ×10⁻⁸ 7.7 × 10⁻⁷ 3.2 × 10⁻⁶ 9.7 × 10⁻⁷ 2.2 × 10⁻⁶ eNOS 9.9 × 10⁻⁸ 5.8 ×10⁻⁷ 2.3 × 10⁻⁶ 2.0 × 10⁻⁴ 2.7 × 10⁻⁵ nNOS 3.1 × 10⁻⁸ 9.0 × 10⁻⁷ 7.9 ×10⁻⁶ 1.5 × 10⁻⁵ 2.8 × 10⁻⁴

In Table 1 is given: IC50 [μM] determination of different compounds forthe different NOS enzymes.

Above mentioned NOS inhibitors are state of the art as depicted below:

-   AMT: Nakane, M. et al., 1995; Tracey, W. R. et al., 1995;    Rairigh, R. L. et al. 1998; Briones, A-M. et al., 1999;-   ODQ: Boulton, C. L. et al., 1995; Brunner, F. et al., 1995;    Garthwaite, J et al., 1995; Moro, M. A. et al., 1996; Schrammel, A.    et al., 1996; Southam, E. et al., 1996; Abi-Gerges, N. et al., 1997;    Olson, L. J, et al., 1997; Sobey C. G. & Faraci F. M. 1997;    Hwang, T. L. et al., 1998; R. Motterlini, et al., 2000;-   L-NMMA: Sakuma, I. et al., 1988; Rees, D. D. et al., 1989;-   1400W: Garvey, E. P et al., 1997; Thomsen, L. L. et al., 1997;    Laszlo F. & Whittle B. J. R, 1997; Gumpricht, E. et al., 2002;-   L-NIL: Moore, W. M. et al., 1994.

Literature

-   Meyer B. and Hemmens B. (1997) Biosynthesis and action of nitric    oxide in mammalian cells Trends Biochem. Sci. 22, 477-481-   Patrick Vallance and James Leiper: Blocking NO Synthesis: How, where    and why? Nature Reviews/Drug Discovery, Volume 1 Dec. 2002, 939-950-   Wendy K. Alderton, Chris E. Cooper and Richard G. Knowles: Nitric    oxide synthases: structure, function and inhibition    Biochem. J. (2001) 357, 593-615-   Janssens S P. Shimouchi A. Quertermous T. Bloch D B. Bloch K D.    Cloning and expression of a cDNA encoding human endothelium-derived    relaxing factor/nitric oxide synthase. Journal of Biological    Chemistry 267(21):14519-22, 1992 Jul. 25.-   Marsden P A. Schappert K T. Chen H S. Flowers M. Sundell C L. Wilcox    J N. Lamas S. Michel T. Molecular cloning and characterization of    human endothelial nitric oxide synthase. FEBS Letters. Vol.    307(3)(pp 287-293), 1992.-   Nakane M. Schmidt HHHW. Pollock J S. Forstermann U. Murad F. Cloned    human brain nitric oxide synthase is highly expressed in skeletal    muscle. FEBS Letters. Vol. 316(2)(pp 175-180), 1993.-   Geller D A. Lowenstein C J. Shapiro R A. Nussler A K. Di Silvio M.    Wang S C. Nakayama D K. Simmons R L. Snyder S H. Billiar T R.    Molecular cloning and expression of inducible nitric oxide synthase    from human hepatocytes. Proceedings of the National Academy of    Sciences of the United States of America. Vol. 90(8)(pp 3491-3495),    1993.-   Sherman P A. Laubach V E. Reep B R. Wood E R. Purification and cDNA    sequence of an inducible nitric oxide synthase from a human tumor    cell line. Biochemistry. Vol. 32(43)(pp 11600-11605), 1993.-   Charles I G. Palmer R MJ. Hickery M S. Bayliss M T. Chubb A P. Hall    V S. Moss D W. Moncada S. Cloning, characterization, and expression    of a cDNA encoding an inducible nitric oxide synthase from the human    chondrocyte. Proceedings of the National Academy of Sciences of the    United States of America. Vol. 90(23)(pp 11419-11423), 1993.-   Maier R. Bilbe G. Rediske J. Lotz M. Inducible nitric oxide synthase    from human articular chondrocytes: cDNA cloning and analysis of mRNA    expression. Biochimica et Biophysica Acta—Protein Structure &    Molecular Enzymology. Vol. 1208(1)(pp 145-150), 1994.-   Chartrain N A. Geller D A. Koty P P. Sitrin N F. Nussler A K.    Hoffman E P. Billiar T R. Hutchinson N I. Mudgett J S. Molecular    cloning, structure, and chromosomal localization of the human    inducible nitric oxide synthase gene. Journal of Biological    Chemistry. Vol. 269(9)(pp 6765-6772), 1994.-   Griffith O W. Stuehr D J. Nitric oxide synthases: Properties and    catalytic mechanism. Annual Review of Physiology. Vol. 57(pp    707-736), 1995-   Mayer, B.: Biochemistry and molecular pharmacology of nitric oxide    synthases, in Nitric Oxide in the Nervous System (S. R. Vincent,    ed.), Academic, New York, 21-42,1995-   Masters B SS. McMillan K. Sheta E A. Nishimura J S. Roman L J.    Martasek P. Neuronal nitric oxide synthase, a modular enzyme formed    by convergent evolution: Structure studies of a cysteine    thiolate-liganded heme protein that hydroxylates L-arginine to    produce NO. as a cellular signal. FASEB Journal. Vol. 10(5)(pp    552-558), 1996.-   Marletta M A. Yoon P S. Iyengar R. Leaf C D. Wishnok J S. Macrophage    oxidation of L-arginine to nitrite and nitrate: Nitric oxide is an    intermediate. Biochemistry. Vol. 27(24)(pp 8706-8711), 1988.-   Kwon N S. Nathan C F. Gilker C. Griffith O W. Matthews D E. Stuehr    D J. L-Citrulline production from L-arginine by macrophage nitric    oxide synthase. The ureido oxygen derives from dioxygen. Journal of    Biological Chemistry. Vol. 265(23)(pp 13442-13445), 1990.-   Mayer B. John M. Heinzel B. Werner E R. Wachter H. Schultz G.    Bohme E. Brain nitric oxide synthase is a biopterin- and    flavin-containing multi-functional oxido-reductase. FEBS Letters.    Vol. 288(1-2)(pp 187-191), 1991.-   Stuehr D J. Kwon N S. Nathan C F. Griffith O W. Feldman P L.    Wiseman J. N(omega)-hydroxy-L-arginine is an intermediate in the    biosynthesis of nitric oxide from L-arginine. Journal of Biological    Chemistry. Vol. 266(10)(pp 6259-6263), 1991.-   Klatt P. Schmidt K. Uray G. Mayer B. Multiple catalytic functions of    brain nitric oxide synthase. Biochemical characterization,    cofactor-requirement, and the role of N(omega)-hydroxy-L-arginine as    an intermediate. Journal of Biological Chemistry. Vol. 268(20)(pp    14781-14787), 1993.-   Feldmann P L. Griffith O W. And Stuehr D J.: The surprising life of    nitric oxide. Chem. Eng. News 71, 26-38, 1993-   Marletta M A. Nitric oxide synthase: Aspects concerning structure    and catalysis. Cell. Vol. 78(6)(pp 927-930), 1994.-   Pufahl R A. Marletta M A. Oxidation of N(G)-hydroxy-L-arginine by    nitric oxide synthase: Evidence for the involvement of the heme in    catalysis. Biochemical & Biophysical Research Communications. Vol.    193(3)(pp 963-970), 1993.-   Clement B. Schultze-Mosgau M-H. Wohlers H. Cytochrome P450 dependent    N-hydroxylation of a guanidine (debrisoquine), microsomal catalysed    reduction and further oxidation of the N-hydroxy-guanidine    metabolite to the urea derivative. Similarity with the oxidation of    arginine to citrulline and nitric oxide. Biochemical Pharmacology.    Vol. 46(12)(pp 2249-2267), 1993.-   Powell D., Williams D. Nitric Oxide Synthase Screening Kit for the    detection of inhibitors of NOS enzyme activity Life Science News,    2002 Amersham Biosiences-   Misko T. P., Schilling R. J., Salvemini D., Moore W. M.,    Currie M. G. A; A Fluorometric Assay for the Measurement of Nitrate    in Biological Samples Analytical Biochemistry 214, 11-16 1993-   Gerzer R., et al.; Soluble guanylate cyclase purified from bovine    lung contains heme and copper; FEBS Lett. 132, 71 (1981)-   Zabel U., et al.; Human soluble guanylate cyclase: functional    expression and revised isoenzyme family; Biochem. J. 335 (Pt 1), 51    (1998)-   Hoenicka, M. et al.; Purified soluble guanylyl cyclase expressed in    a baculovirus/Sf9 system: stimulation by YC-1, nitric oxide, and    carbon monoxide; J. Mol. Med. 77, 14 (1999)-   Lee, Y. C. et al.; Human recombinant soluble guanylyl cyclase:    expression, purification, and regulation; PNAS 97, 10763 (2000)-   Kosarikov, D. N. et al.; Human soluble guanylate cyclase: functional    expression, purification and structural characterization; Arch.    Biochem. Biophys. 388, 185 (2001) Claret E. J., et al.; A new HTRF    cGMP assay for monitoring Guanylyl cyclase activity; Poster    presentation at the SBS Meeting in Orlando (2004)-   Perkin Elmer: Flashplate cyclic cGMP [¹²⁵I]-assay product no:    SMP002J001PK Keele, B. B. Jr. et al.; Superoxide Dismutase from    Escherichia coli B; JBC Vol.245, No. 22, pp 6176-6181 (1970).-   Beckman J. S. et al.; Apparent hydroxyl radical production by    peroxynitrite: Implications for endothelial injury from nitric oxide    and superoxide; Proc. Natl. Acad. Sci USA Vol 87, pp. 1620-1624    (1990).-   Drago R. S. & Paulik F. E.; The Reaction of Nitrogen(II) Oxide with    Diethylamine; JACS 82, 96 (1960)-   Drago R. S. & B. R. Karstetter B. R.; The Reaction of Nitrogen(II)    Oxide with Various Primary and Secondary Amines; JACS 83, 1819    (1961)-   Maragos, C. M. et al.; Complexes of NO with nucleophiles as agents    for the controlled biological release of nitric oxide. Vasorelaxant    effects; J. Med. Chem. 34, 3242 (1991)-   Wink D. A., et al.; DNA deaminating ability and genotoxicity of    nitric oxide and its progenitors; Science 254, 1001 (1991)-   Hrabie, et al.; New nitric oxide-releasing zwitterions derived from    polyamines; JOC 58, 1472 (1993)-   Robak, et al.; Pharmacol. Res. 25 S2, 355 (1992)-   Moilanen, E: et al.; Inhibition by nitric oxide-donors of human    polymorphonuclear leucocyte functions; Br. J. Pharmacol. 109, 852    (1993)-   Corell, T. et al.; Pharmacology of mesoionic oxatriazole derivatives    in blood, cardiovascular and respiratory systems; Pol. J. Pharmacol.    46, 553 (1994)-   Karup, G. et al.; Mesoionic oxatriazole derivatives—a new group of    NO-donors; Pol. J. Pharmacol. 46, 541 (1994)-   Malo-Ranta, U. et al.; Nitric oxide donor GEA 3162 inhibits    endothelial cell-mediated oxidation of low density lipoprotein; FEBS    Lett. 337, 179 (1994)-   Ma, H. T. et al.; Ca(2+) entry activated by S-nitrosylation.    Relationship to store-operated ca(2+) entry; J. Biol. Chem. 274,    35318 (1999)-   Nakane, M. et al.; Novel potent and selective inhibitors of    inducible nitric oxide synthase; Mol. Pharmacol. 47, 831 (1995)-   Tracey, W. R. et al.; In vivo pharmacological evaluation of two    novel type II (inducible) nitric oxide synthase inhibitors; Can. J.    Physiol. Pharmacol. 73, 665 (1995)-   Rairigh, R. L. et al.; Role of inducible nitric oxide synthase in    regulation of pulmonary vascular tone in the late gestation ovine    fetus; J. Clin. Invest. 101, 15 (1998)-   Briones, A-M. et al.; Role of iNOS in the vasodilator responses    induced by L-arginine in the middle cerebral artery from    normotensive and hypertensive rats; Br. J. Pharmacol. 126, 111    (1999)-   Boulton, C. L. et al.; Nitric oxide-dependent long-term potentiation    is blocked by a specific inhibitor of soluble guanylyl cyclase;    Neuroscience 69, 699 (1995)-   Brunner, F. et al.; Novel guanylyl cyclase inhibitor, ODQ reveals    role of nitric oxide, but not of cyclic GMP in endothelin-1    secretion; FEBS Lett. 376, 262 (1995)-   Garthwaite, J et al.; Potent and selective inhibition of nitric    oxide-sensitive guanylyl cyclase by    1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; Mol. Pharmacol. 48, 184    (1995)-   Moro, M. A. et al.; cGMP mediates the vascular and platelet actions    of nitric oxide: confirmation using an inhibitor of the soluble    guanylyl cyclase; PNAS 93, 1480 (1996)-   Schrammel, A. et al.; Characterization of    1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one as a heme-site inhibitor    of nitric oxide-sensitive guanylyl cyclase; Mol. Pharmacol. 50, 1    (1996)-   Southam, E. et al.; The nitric oxide-cyclic GMP pathway and synaptic    plasticity in the rat superior cervical ganglion; Br. J. Pharmacol.    119, 527 (1996)-   Abi-Gerges, N. et al.; A comparative study of the effects of three    guanylyl cyclase inhibitors on the L-type Ca2+ and muscarinic K⁺    currents in frog cardiac myocytes; Br. J. Pharmacol. 121, 1369    (1997)-   Olson, L. J, et al.; Selective guanylyl cyclase inhibitor reverses    nitric oxide-induced vasorelaxation; Hypertension 29, 254 (1997)    Sobey C. G. & Faraci F. M.; Effects of a novel inhibitor of guanylyl    cyclase on dilator responses of mouse cerebral arterioles; Stroke    28, 837 (1997)-   Hwang, T. L. et al.; Comparison of two soluble guanylyl cyclase    inhibitors, methylene blue and ODQ, on sodium nitroprusside-induced    relaxation in guinea-pig trachea; Br. J. Pharmacol 125, 1158 (1998)-   Motterlini, R et al.; Endothelial heme oxygenase-1 induction by    hypoxia. Modulation by inducible nitric-oxide synthase and    S-nitrosothiols; J. Biol. Chem. 275, 13613 (2000)-   Sakuma, I. et al.; Identification of arginine as a precursor of    endothelium-derived relaxing factor; PNAS 85, 8664 (1988)-   Rees, D. D. et al.; A specific inhibitor of nitric oxide formation    from L-arginine attenuates endothelium-dependent relaxation; Br. J.    Pharmacol. 96, 418 (1989)-   Garvey, E. P et al.; 1400W is a slow, tight binding, and highly    selective inhibitor of inducible nitric-oxide synthase in vitro and    in vivo; J. Biol. Chem. 272, 4959 (1997)-   Thomsen, L. L. et al.; Selective inhibition of inducible nitric    oxide synthase inhibits tumor growth in vivo: studies with 1400W, a    novel inhibitor; Cancer Res. 57, 3300 (1997)-   Laszlo F. & Whittle B. J. R.; Actions of isoform-selective and    non-selective nitric oxide synthase inhibitors on endotoxin-induced    vascular leakage in rat colon; Eur. J. Pharmacol. 334, 99 (1997)-   Gumpricht, E. et al.; Nitric Oxide Ameliorates Hydrophobic Bile    Acid-induced Apoptosis in Isolated Rat Hepatocytes by    Non-mitochondrial Pathways; J. Biol. Chem. 277, 25823 (2002)-   Moore, W. M. et al.; L-N-6-(1-iminoethyl)lysine: a selective    inhibitor of inducible nitric oxide synthase; J. Med. Chem. 37, 3886    (1994)    Description of FIG. 1:

FIG. 1 shows a Dose response testing of the synthetic NO donor DEANONOate on the stimulation of soluble guanylyl cyclase and detection ofthe subsequent cGMP production of guanylyl cyclase by employing theAlphaScreen cGMP competition assay outlined in Example 4.

1. A method for measuring nitric oxide synthase activity, wherein themethod comprises: (a) providing a solution comprising a nitric oxidesynthase and a suitable substrate under conditions which allow synthesisof nitric oxide; (b) adding guanylyl cyclase and GTP under conditionswhich allow synthesis of cGMP; and (c) measuring cGMP, thereby measuringnitric oxide synthase activity.
 2. The method according to claim 1,wherein the substrate is arginine.
 3. The method according to claim 1,wherein cGMP is measured by an anti-cGMP specific antibody.
 4. Themethod according to claim 1, wherein the nitric oxide synthase is i-NOS,e-NOS, or n-NOS.
 5. A method for measuring nitric oxid synthase (NOS)activity in an high throughput (HTS) format wherein the method accordingto claim 1 is performed.
 6. The method according to claim 5 wherein aplate having at least 96 wells is used.
 7. The method according to claim6 wherein a 384 well plate or a 1536 well plate is used.
 8. The methodaccording to claim 5 wherein a chip is used as a reaction and/or readoutplatform.
 9. The method according to claim 1, wherein superoxidedismutase is added in step (b) together with guanylyl cyclase and GTP.10. The method according to claim 1,wherein the method comprises: (a)providing a solution comprising a nitric oxide synthase, superoxidedismutase and a suitable substrate under conditions which allowsynthesis of nitric oxide; (b) adding guanylyl cyclase and GTP underconditions which allow synthesis of cGMP; and (c) measuring cGMP,thereby measuring nitric oxide synthase activity.
 11. The methodaccording to claim 10 wherein the substrate is arginine.
 12. The methodaccording to claim 11 wherein cGMP is measured by an anti- cGMP specificantibody.
 13. The method according to claim 11 wherein the nitric oxidesynthase is i-NOS, e-NOS, or n-NOS.
 14. A method for measuring nitricoxid synthase (NOS) activity in an high throughput (HTS) format whereinthe method according to claim 10 is performed.
 15. The method accordingto claim 14 wherein a plate having at least 96 wells-is used.
 16. Themethod according to claim 15 wherein a 384 well plate or a 1536 wellplate is used.
 17. The method according to claim 14 wherein a chip isused as a reaction and/or readout platform.